Methods for introducing mannose 6-phosphate and other oligosaccharides onto glycoproteins and applications thereof

ABSTRACT

Methods to introduce highly phosphorylated mannopyranosyl oligosaccharide derivatives containing mannose-6-phosphate (M6P), or other oligosaccharides bearing other terminal hexoses, to carbonyl groups on oxidized glycans of glycoproteins while retaining their biological activity are described. The methods are useful for modifying glycoproteins, including those produced by recombinant protein expression systems, to increase uptake by cell surface receptor-mediated mechanisms, thus improving their therapeutic efficacy in a variety of applications.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/463,955, filed Aug. 20, 2014, which is a continuation of U.S. patentapplication Ser. No. 13/768,266 (now U.S. Pat. No. 8,841,427), filedFeb. 15, 2013, which is a continuation of U.S. patent application Ser.No. 12/642,383 (now U.S. Pat. No. 8,399,657), filed Dec. 18, 2009, whichis a continuation of U.S. patent application Ser. No. 10/943,893 (nowU.S. Pat. No. 7,723,296), filed Sep. 20, 2004, which is acontinuation-in-part of U.S. patent application Ser. No. 10/051,711 (nowU.S. Pat. No. 7,001,994), filed Jan. 17, 2002, which claims the benefitof priority of U.S. Provisional Application No. 60/263,078, filed Jan.18, 2001, the disclosures of which are incorporated herein by referencein their entirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name:159792013704SUBSEQLIST.TXT, date recorded: Jul. 11, 2017, size: 12 KB).

BACKGROUND OF THE INVENTION

The present invention relates in general to methods for introducing newoligosaccharides to glycoproteins, and more specifically, to novelmethods for conjugating highly phosphorylated mannopyranosyloligosaccharide derivatives to glycoproteins to form compoundscontaining mannose-6-phosphate (M6P) for use in medical methods oftreatment, and to the compounds thereby produced.

Carbohydrates on glycoproteins play important biological functions inbio-organisms. Well-characterized examples include theselectin-carbohydrate interaction involved in Intercellular celladhesion and sperm/egg interaction (see, e.g., C. G. Gahmberg et al., 27APMIS SUPPL. 39 (1992)), and the mannose 6-phosphate (M6P) dependentlysosomal enzyme targeting pathway (see, e.g., S. Kornfeld and I.Mellman, 5 ANNUAL REVIEW OF CELL BIOLOGY 483 (1989)). To facilitatestudy of the complex functions of carbohydrate structures onglycoproteins, both enzymatic and chemical methods have been developedto remove the carbohydrate glycans from glycoproteins for analysis. Avariety of conjugation methods have also been developed to conjugatedefined carbohydrates to proteins and then analyze their possiblebiological functions. The most commonly used conjugation approachinvolves the use of omega-amino groups of lysine residues. Reactions ofamino groups of proteins with compounds such as N-hydroxysuccinimideester or isothiocyanate derivatives are widely used. Reductiveamination, on the other hand, is most commonly used for carbohydrateconjugation to proteins. For example, in the analysis of lysosomalenzyme targeting, coupling of M6P or phosphopentamannose to bovine serumalbumin has been achieved through reductive amination (H. Tomoda et al.,213 CARBOHYDR. RES. 37 (1991); T. Baba et al., 177 CARBOHYDR. RES. 163(1988)), leading to significant insights regarding the M6P receptorbinding to lysosomal enzymes through these M6P conjugated glycoproteins.Reductive amination involves covalently linking the reducing ends ofoligosaccharides to amino acid residues in proteins containing freeamines (such as lysines), to first form unstable Schiff bases which arethen reduced by cyanoborohydride to stable imine bonds.

However, these known conjugation methods are limited in that they arenot specific in terms of the amino acid residues involved, and requirethe direct linkage of chemical conjugates or carbohydrates to amino acidresidues, which may cause a change in protein conformation and destroythe biological activity of proteins. For example, when antibody IgG iscoupled to various chemical conjugates through amino acid groups, theantibody IgG often loses its immunological activity (D. J. O'Shannessyand R. H. Quarles, 7 J. OF APPL. BIOCHEM. 347, (1985); T. I. Ghose etal., 93 METHODS IN ENZYMOLOGY 280 (1983)). In addition, reductiveamination requires high pH and a reductive reagent that may also reduceany disulfide bonds in a protein, thus potentially destroying biologicalactivity.

A more specific approach to introduce certain chemical conjugates ontoglycoproteins has been described and involves covalent bond formationbetween carbonyl (aldehyde) groups generated by mild oxidation ofcarbohydrates with periodate or galactose oxidase (G. Avigad et al., 237J. BIOL. CHEM. 2736 (1962)) and chemical compounds containingcarbonyl-reactive groups. This approach has been used for in vitroattachment of mono- and oligosaccharides to cell surface glycoconjugatesof living cells with glycosylhydrazines (M. Tolvanen and C. G. Gahmberg,261(2) J. BIOL. CHEM. 9546 (1986); C. G. Gahmberg and M. Tolvanen, 230METHODS IN ENZYMOLOGY 32 (1994)). Other applications include conjugationof biotin or avidin to glycoproteins with biotin-hydrazide oravidin-hydrazide (Bayer et al., 161 ANALYTICAL BIOCHEMISTRY 123 (1987);Bayer et al., 170 ANALYTICAL BIOCHEMISTRY 271 (1988); M. Wilchek and E.A. Bayer, 128 METHODS IN ENZYMOLOGY 429 (1987)), antibody IgGconjugation for immunodetection (O'Shannessy et al., 8 IMMUNOLOGYLETTERS 273 (1984); D. J. O'Shannessy and R. H. Quarles, 7 J. OF APPL.BIOCHEM. 347 (1985)) and cancer immunotherapy (J. Singh Kralovec et al.,29 CANCER IMMUNOLOGY THERAPY 293 (1989); G. R. Braslawsky et al., 33CANCER IMMUNOLOGY THERAPY 367 (1991)). In these examples, theglycoproteins are treated by mild oxidation with periodate to generatealdehyde groups that then react with hydrazide derivatives. Oneadvantage of this approach for conjugation is that the linkage isthrough the carbohydrates on the glycoproteins instead of directlyinvolving the protein backbone, thus avoiding inactivation of theglycoproteins' biological activity. Antibodies modified in such a wayalways retain activity (D. J. O'Shannessy et al., 8 IMMUNOLOGY LETTERS273 (1984); D. J. O'Shannessy and R. H. Quarles, 7 J. OF APPL. BIOCHEM.347 (1985)). In addition, both the oxidation and the covalent bondformation steps are nearly quantitative, and reaction conditions arevery mid, thus helping retain the biological activity of the proteins.Retention of biological activity is critical when the modifiedglycoproteins are to be used for therapeutic purposes.

Lysosomal storage disease describes a class of over 40 genetic disorders(see, e.g., Holton, J. B., THE INHERITED METABOLIC DISEASES 205-242(2^(nd) ed. 1994); Scriver et al., 2 THE METABOLIC BASIS OF INHERITEDDISEASE (7^(th) ed. 1995)), each resulting from a deficiency of aparticular lysosomal enzyme, usually as a result of genetic mutation.Lysosomal enzymes are required to break down protein, glycolipid andcarbohydrate metabolites within the lysosomes of cells. When one or moreof these enzymes are defective in affected individuals due to inheritedmutations, lysosomes in cells of affected individuals accumulate asubset of undigested substrates, largely liposaccharides andcarbohydrates as storage materials that are unable to be digested by thedefective enzymes. For example, in Gaucher disease, deficiency ofbeta-glucocerebrosidase causes the accumulation of glucosylceramide; inFabry disease, the defective alpha-galactosidase A results inaccumulation of globotriaosylceremide; in Pompe disease, lack of acidalpha-glucosidase causes accumulation of glycogen alpha 1-4 linkedoligosaccharides and in Tay-Sachs disease, deficiency ofbeta-N-acetyl-hexosaminidase leads to accumulation of GM2 ganglioside.Clinically, patients with these syndromes show a variety of symptomsassociated with the accumulation of these storage material in thelysosomes, which eventually affect the normal function of the cells ortissues that result in dysfunction of organs within the human body. Theseverity of the disease varies with the residual enzyme activity, insevere cases, death can occur early in life.

Lysosomal enzymes, like other secretory proteins, are synthesized andco-translationally translocated into the lumen of the endoplasmicreticulum, where post-translational carbohydrate modification occurs.However, while in transit through the Golgi, they are segregated fromthe other secretory proteins by specifically acquiring the M6Precognition marker generated by the sequential actions of two enzymes.The first enzyme, UDP-N-acetylglucosamine:Lysosomal-enzymeN-Acetylglucosamine-1-phosphotransferase, transfers theN-acetylglucosamine-1-phosphate to one or more mannose residues onlysosomal enzymes to give rise to phophodiester intermediates, and thesecond enzyme, N-acetylglucosamine-1-phosphodiesteralpha-N-acetylglucosaminidase, removes the N-acetylglucosamine from thephosphodiester to expose the M6P. Once the lysosomal enzymes with theM6P recognition marker reach the trans-Golgi-network, they arerecognized by two specific receptors, the cation-independent mannose6-phosphate receptor (CI-MPR) and the cation-dependent mannose6-phosphate receptor (CD-MPR). These receptors with their ligands oflysosomal enzymes are sequestered into clathrin-coated vesicles formedon the trans-Golgi network and transported to endosomes, where thelysosomal enzymes are dissociated from the receptors by the low pH inendosomes and eventually delivered to lysosomes. Some of the lysosomalenzymes are secreted, however, they are captured by binding to theCI-MPR on the cell surface and internalized by the AP-2 mediatedclathrin-coated vesicles. Thus, the M6P dependent pathway is the maintargeting pathway for lysosomal enzymes, though the M6P independenttargeting pathways have been proposed for a few lysosomal enzymes and incertain cell types (see Kornfeld and Mellman, supra).

With the complete elucidation of the lysosomal enzyme targeting pathwayand the discovery of lysosomal enzyme deficiencies as the primary causeof lysosomal storage diseases, attempts have been made to treat patientshaving lysosomal storage diseases by intravenous administration of themissing enzyme, i.e., enzyme replacement therapy, where the injectedenzymes are expected to be taken up by target cells throughreceptor-mediated endocytosis and delivered to lysosomes. Animal modelsand some clinical trials of enzyme replacement therapy have offeredpositive results. However, for lysosomal diseases other than Gaucherdisease, some evidence suggest that enzyme replacement therapy is mosteffective when the enzyme being administered has M6P, so that theenzymes can be taken up efficiently by the target cells through the cellsurface associated CI-MPR-mediated endocytosis. Gaucher disease, causedby the deficiency of beta-glucocerebrosidase, is an exception becausebeta-glucocerebrosidase is among the few lysosomal enzymes that aretargeted by the M6P independent pathway (see Kornfeld and Mellman,supra). Targeting of beta-glucocerebrosidase for Gaucher disease enzymereplacement therapy to macrophage cells is mediated by remodeling itscarbohydrate to expose the core mannose, which binds to the mannosereceptor on macrophage cell surface.

While enzyme replacement therapy (ERT) appears promising, supplies ofthe required enzymes are limited. Lysosomal enzymes can, in theory, beisolated from natural sources such as human placenta or other animaltissues. However, large-scale production of sufficient quantities ofenzymes for therapeutic administration is difficult. Further, due to thedegradation of carbohydrates in lysosomes, enzymes purified from tissuesdo not contain significant amounts of M6P. Alternative approachesinclude use of recombinant protein expression systems, facilitated bylarge-scale cell culture or fermentation. For example, lysosomal enzymeshave been expressed in Chinese hamster ovary (CHO) cells (V. A. Ioannouet al., 119(5) J. CELL BIOL. 1137 (1992); E. D. Kakkis et al., 5 PROTEINEXPRESSION PURIFICATION 225 (1994)), insect cells (V. Chen et al., 20(2)PROTEIN EXPR. PURIF. 228 (2000)), and in transgenic animals or plants(A. G. Bijvoet et al., 8(12) HUM. MOL. GENET. 2145 (1999)). However,lysosomal enzymes purified from recombinant expression systems are alsooften not well phosphorylated and the extent of M6P phosphorylationvaries considerably with different enzymes. Alpha-galactosidase Aexpressed in CHO cells contains about 20% of phosphorylated enzymes, butonly 5% are bisphosphorylated, which is the high-uptake form (F.Matsuura et al., 8(4) GLYCOBIOLOGY 329 (1998)).Alpha-N-acetylglucosaminidase expressed in CHO cells is almostcompletely lacking M6P phosphorylation (K. Zhao and E. F. Neufeld, 19PROTEIN EXPR. PURIF. 202 (2000)). In addition, recombinant proteinsexpressed in plants, insect cells or the methotrophic yeast pichiapastoris do not have any M6P phosphorylation because such cells do nothave the M6P targeting pathway.

Lysosomal enzymes lacking in M6P phosphorylation compete poorly forreceptor-mediated endocytic uptake by target cells and are thus oflimited efficacy in enzyme replacement therapy. More specifically,poorly phosphorylated enzymes are effectively removed by the mannosereceptor (M. E. Taylor et al., 252 AM. J. PHYSIOL. E690 (1987)) andasiologlycoprotein receptor in liver (Ashwell and Harford, 51 ANN. REV.BIOCHEM 531 (1982)), which can remove most of any administered lysosomalenzymes within a very short period of time.

Against this background, a strong need exists for improved, efficientapproaches to phoshorylate lysosomal enzymes, and particularly formethods to modify lysosomal enzymes with M6P. In addition, a need existsfor modifying lysosomal enzymes to a high uptake, bisphosphorylatedform. Such modified enzymes would be particularly useful for enhancingthe efficacy of enzyme replacement therapy for lysosomal storagedisease.

BRIEF SUMMARY OF THE INVENTION

Methods of creating neoglycoproteins are provided that increase thecellular uptake of lysosomal enzymes and other glycoproteins bycovalently attaching oligosaccharide compositions to oxidized glycans ofthe glycoproteins through covalent bonds.

Thus, in one embodiment, the invention is directed toward a method forcoupling a highly phosphorylated mannopyranosyl oligosaccharide compoundto a glycoprotein having at least one glycan, the method comprisingderivatizing the highly phosphorylated mannopyranosyl oligosaccharidecompound with a chemical compound containing a carbonyl-reactive group;oxidizing the glycoprotein having the at least one glycan to generate atleast one aldehyde group on the glycoprotein; and reacting the oxidizedglycoprotein having at least one glycan with the derivatized highlyphosphorylated mannopyranosyl oligosaccharide compound to form a newcompound having a hydrazone bond. The glycoprotein in one embodiment isa lysosomal enzyme.

In one embodiment of the methods, the highly phosphorylatedmannopyranosyl oligosaccharide compound contains at least one mannose6-phosphate group, such as a compound having the formula 6-P-M_(n)-Rwherein:

M is a mannose or mannopyranosyl group;

P is a phosphate group linked to the C-6 position of M;

R comprises a chemical group containing at least one carbonyl-reactivegroup; and

n is an integer from 1-15, wherein if n>1, M_(n) are linked to oneanother by alpha (1,2), alpha (1,3), alpha (1,4), or alpha (1,6).

Thus, the highly phosphorylated mannopyranosyl oligosaccharide compoundincludes compounds such as M6P, phosphopentamannose derived fromHansenula holstii O-phosphomannan, and 6-P-M-(alpha 1,2)-M(alpha 1,2)-M.

In another embodiment of the methods, the highly phosphorylatedmannopyranosyl oligosaccharide compound comprises a compound having theformula (6-P-M_(x))_(m)L_(n)-R wherein:

M is a mannose or mannopyranosyl group;

L is a mannose or other hexose or other chemical groups;

P is a phosphate group linked to the C-6 position of M;

R comprises a chemical group containing at least one carbonyl-reactivegroup;

m is an integer from 2-3;

n is an integer from 1-15, wherein if n>1, M_(n) are linked to oneanother by alpha (1,2), alpha (1,3), alpha (1,4), or alpha (1,6); and

x is an integer from 1-15.

Thus, the highly phosphorylated mannopyranosyl oligosaccharide compoundincludes biantennary mannopyranosyl oligosaccharide compounds containingbis-M6P and triantennary mannopyranosyl oligosaccharide compoundscontaining bis-M6P or tri-M6P.

In one embodiment of the methods, the highly phosphorylatedmannopyranosyl oligosaccharide compound can be replaced with otheroligosaccharide compositions containing terminal hexoses, such as, forexample, a galactose, a mannose, N-acetylglucosamine, or a fucose, whichcan bind to different carbohydrate-binding receptors other than CI-MPR.

In another embodiment of the methods, the chemical compound containingcarbonyl-reactive group includes any compound that reacts with carbonylgroups to form a hydrazone bond. Such compounds include hydrazines,hydrazides, aminooxyls, and semicarbozides and the like.

In addition, the methods further encompass reducing the compound havinga hydrazone bond with a reducing agent such as cyanoborohydride to forma compound having an imine bond.

The invention is further directed toward chemical compounds produced bycoupling a first chemical compound having at least one carbonyl group(aldehyde or ketone) to a second chemical compound comprising aphosphorylated mannopyranosyl oligosaccharide derivative, according tothe coupling methods described and herein, i.e., by derivatizing thehighly phosphorylated mannopyranosyl oligosaccharide compound with achemical compound containing a carbonyl-reactive group; and reacting tothe first chemical compound having at least one carbonyl group with thederivatized highly phosphorylated mannopyranosyl oligosaccharidecompound to form a new compound having a hydrazone bond. Such compoundsinclude antiviral compounds and gene targeting delivery agents.

In another embodiment, the invention is directed toward methods oftreating lysosomal storage disease in a subject in need thereof, themethods including administering to the subject an effective amount of aglycoprotein coupled according to the methods described herein to asecond chemical compound comprising a highly phosphorylatedmannopyranosyl oligosaccharide derivative containing at least onemannose 6-phosphate group. Lysosomal storage diseases that are treatedwith a glycoprotein modified according to the methods described hereininclude Fabry disease, Pompe disease, and others (for a complete list,see J. B. Holton, THE INHERITED METABOLIC DISEASES 205-242 (2d ed.1994); C. R. Scriver et al., 2 THE METABOLIC BASIS OF INHERITED DISEASE(7^(th) ed. 1995)).

The present methods couple highly phosphorylated mannopyranosyloligosaccharides containing M6P, to glycoproteins, so that cellularuptake of such glycoproteins is enhanced without destroying theirbiological activity. As such, the methods and compounds produced therebyare especially useful where in medical treatment methods that benefitfrom enhanced uptake forms of glycoproteins, such as in enzymereplacement therapy for the treatment of lysosomal storage diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of a conjugation method.

FIGS. 2A-2C are schematic representations of alternative oxidationmethods used in the conjugation methods.

FIG. 3 is a SDS-PAGE analysis of different avidins before and afterphosphopentamannose-hydrazine conjugation.

FIG. 4 is a CI-MPR binding analysis of untreated avidin and oxidizedavidin conjugated with phosphopentamannose-hydrazine.

FIG. 5 is a bar graph showing enzymatic activity of beta-glucuronidaseafter different treatments including after conjugation.

FIG. 6 is a bar graph comparing CI-MPR binding of untreated and oxidizedphosphopentamannose-conjugated beta-glucuronidase.

FIG. 7 depicts GAA activity, measured in nmole/hour, in fractionsobtained from a CI-MPR column following binding of recombinant human GAA(shown by open circles), and phosphopentamannose-conjugated rhGAA (shownby closed circles) to the CI-MPR column.

FIG. 8A depicts the results of a chromatographic analysis ofoligosaccharides released following digestion of α-galactosidase A withendoglycosidase H. FIG. 8B depicts the results of a chromatographicanalysis of oligosaccharides following purification of theM6P-containing fraction over a QAE column by dionex columnchromatography.

FIG. 9 depicts GAA activity in fractions obtained following binding ofrhGAA (shown by open circles), modified rhGAA (neo-rhGAA) (shown byclosed circles) and periodate-treated rhGAA (shown by open squares) to aCI-MPR column.

FIG. 10 depicts a graph showing the in vitro uptake of the modifiedrhGAA (neo-rhGAA) into L6 myoblasts. Open circles represent increasingamounts of rhGAA and closed squares represent increasing amounts ofmodified rhGAA (neo-rhGAA). Endogenous GAA levels were subtracted fromthe data presented.

FIGS. 11A-11D depict graphs representing the level of glycogen in heart(11C), skeletal muscles (Quadriceps in 11A; Triceps in 11B), anddiaphragm (11D) of Pompe mice treated with varying doses of either rhGAAor neo-rhGAA.

FIG. 12 depicts a bar graph showing the percentage of area occupied byglycogen in quadriceps muscle of Pompe mice treated with vehicle control(A); 10 mg/kg rhGAA (B); 50 mg/kg rhGAA (C); and 20 mg/kg neo-rhGAA (D).

FIG. 13A is a schematic representation of the naturally occurring highmannose oligosaccharide (Man9) from which the synthetic bis-M6P glycan,shown in FIG. 13B, is derived.

FIG. 14A depicts GAA activity of the unmodified rhGAA and modifiedneo-rhGAA, conjugated with synthetic bis-M6P. FIG. 14B depicts GAAactivity in fractions obtained following binding of rhGAA (open circles)and synthetic bis-M6P conjugated rhGAA (closed circles) to a CI-MPRcolumn.

FIGS. 15A and 15B depict results of in vitro uptake assays in L6myoblasts and macrophages, respectively. FIG. 15A shows in vitro uptakeof rhGAA (closed circles) and neo-rhGAA (closed squares) into L6myoblasts, as measured by GAA activity in cell lysates followingtreatment with rhGAA or neo-rhGAA. FIG. 15B depicts a bar graph showingthe results of an in vitro uptake experiment in macrophages, treatedeither with rhGAA or neo-rhGAA, and further in presence or absence ofM6P and/or mannan. As in the case of myoblasts, in vitro uptake wasmeasured by assaying for GAA activity in cell lysates.

FIG. 16 is a bar graph showing glycogen levels, as assayed using theAmplex Red glucose assay, in representative tissues of young Pompe mice(4-5 months of age) following treatment with varying doses of vehiclealone, unmodified rhGAA or neo-rhGAA.

FIG. 17 is a bar graph showing glycogen levels, as assayed using theAmplex Red glucose assay, in representative tissues of old Pompe mice(13 months of age) following treatment with vehicle alone, 40 mg/kg ofunmodified rhGAA or 40 mg/kg of neo-rhGAA.

DETAILED DESCRIPTION OF THE INVENTION

The present methods couple highly phosphorylated mannopyranosyloligosaccharides containing M6P to glycoproteins, such as, for example,avidin and lysosomal enzymes beta-glucuronidase and acidalpha-glucosidase, without destroying their biological activity. Thepresent methods thus provide a novel approach to introduce highlyphosphorylated mannosyloligosaccharide derivatives to lysosomal enzymesand other glycoproteins. In exemplary embodiments, the methods andcompounds described herein are useful for modifying lysosomal enzymesproduced by recombinant protein expression system with M6P, thus toenhance the efficacy of enzyme replacement therapy of lysosomal storagediseases.

As used herein, the term “highly phosphorylated” refers to acharacteristic of oligosaccharides that are coupled to glycoproteins orto other compounds according to the methods described herein, whereinthe oligosaccharides contain at least one M6P group and, in an exemplaryembodiment, two or more M6P groups.

As used herein, the term “effective” refers to a characteristic of anamount of a compound produced according to the methods of the presentinvention, wherein the amount of the compound has the effect ofpreventing or reducing a deficiency of a lysosomal enzyme in a subject.The lysosomal enzyme deficiency is, for example, the result of a geneticmutation in a human that produces a lysosomal storage disease. Suchdiseases include, for example, Gaucher disease wherein a deficiency ofbeta-glucocerebrosidase results in the accumulation of glucosylceramide,Fabry disease wherein a deficiency of alpha-galactosidase A results inaccumulation of globotriaosylceremide, Pompe disease wherein adeficiency of acid alpha-glucosidase results in accumulation of glycogenalpha 1-4-linked oligosaccharides, and Tay-Sachs disease wherein adeficiency of beta-N-acetyl-hexosaminidase leads to accumulation of GM2ganglioside, and other diseases including Hurler or Hurler-Scheledisease, Krabbe disease, Metachromatic leukodystrophy, Hunter disease,Sanfilippo A and B disease, Morquip A disease, and Maroteaux-Lamydisease and other diseases (see Holton, J. B., 1994, THE INHERITEDMETABOUC DISEASES, 2^(nd) Edition; Scriver et al., 1995, THE METABOLICBASIS OF INHERITED DISEASE, Volume 2, 7^(th) Edition, which are hereinincorporated by reference).

Thus, in an exemplary embodiment, a method for coupling a highlyphosphorylated mannopyranosyl oligosaccharide compound to a glycoproteinhaving at least one glycan includes derivatizing the highlyphosphorylated mannopyranosyl oligosaccharide compound with a chemicalcompound containing a carbonyl-reactive group; oxidizing theglycoprotein having the at least one glycan to generate at least onealdehyde group on the glycoprotein; and reacting the oxidizedglycoprotein with the derivatized highly phosphorylated mannopyranosyloligosaccharide compound to form a new compound having a hydrazone bond.Oxidizing the glycoprotein having the at least one glycan isaccomplished using, for example, periodate or galactose oxidase.

The glycoprotein having the at least one glycan is, for example, aglycoprotein such as a lysosomal enzyme. The glycoprotein can be derivedfrom a variety of sources. In the case of lysosomal enzymes, naturalsources include human placenta and other animal tissues. Alternatively,lysosomal enzymes that are especially useful for modification accordingto the present methods are produced by recombinant protein expressionsystems, including yeast, mammalian cells, insect cells, plant cells andtransgenic animals or plants.

The chemical compound containing the carbonyl-reactive group is anycompound that reacts with carbonyl groups to form a hydrazone bond.Suitable such compounds include, for example, hydrazine, hydrazide,aminooxyl, and semicarbozide and the like.

In one embodiment, the highly phosphorylated mannopyranosyloligosaccharide compound contains at least one mannose 6-phosphategroup, such as an oligosaccharide of the formula 6-P-M_(n)-R wherein:

M is a mannose or mannopyranosyl group;

P is a phosphate group linked to the C-6 position of M;

R comprises a chemical group containing at least one carbonyl-reactivegroup; and

n is an integer from 1-15, wherein if n>1, M_(n) are linked to oneanother by alpha (1,2), alpha (1,3), alpha (1,4), or alpha (1,6).

Thus, the highly phosphorylated mannopyranosyl oligosaccharide compoundincludes compounds such as M6P, phosphopentamannose derived fromHansenula holstii O-phosphomannan, and 6-P-M-(alpha 1,2)-M(alpha 1,2)-M.

In an exemplary embodiment, the oligosaccharides are those biantennaryand triantennary oligosaccharides that have the formula of(6-P-M_(x))_(m)L_(n)-R wherein:

M is a mannose or mannopyranosyl group;

L is a mannose or other hexose or other chemical groups;

P is a phosphate group linked to the C-6 position of M;

R comprises a chemical group containing at least one carbonyl-reactivegroup;

m is an integer from 2-3;

n is an integer from 1-15, wherein if n>1, M_(n) are linked to oneanother by alpha (1,2), alpha (1,3), alpha (1,4), or alpha (1,6); and

x is an integer from 1-15.

Thus, the highly phosphorylated mannopyranosyl oligosaccharide compoundincludes biantennary mannopyranosyl oligosaccharide compounds containingbis-M6P and triantennary mannopyranosyl oligosaccharide compoundscontaining bis-M6P or tri-M6P. An exemplary such compound is

-   -   6-P-M(alpha 1,2)-M(alpha 1,3)-        -   M    -   6-P-M(alpha 1,2)-M(alpha 1,6)-

which has about 100 times higher affinity to the MPRs than thephosphopentamannose and M6P, and about 10 times higher affinity to theMPRs than the bi- or tri-oligosaccharides bearing a terminal M6P(Distler et al. 1991).

Alternatively, the highly phosphorylated mannopyranosyl oligosaccharidecompound can be replaced with oligosaccharides containing terminalhexoses, such as a galactose, a mannose, N-acetylglucosamine, or afucose, which can bind to different carbohydrate-binding receptors otherthan CI-MPR.

In addition, methods include the further step of reducing a compoundhaving a hydrazone bond with a reducing agent to form a compound havingan imine bond, which is more stable than the hydrazone bond. Thereducing agent is, for example, a cyanoborohydride compound.

FIG. 1 is a schematic representation of the conjugation methods. In afirst step, the reducing terminal sugar of oligosaccharides isderivatized to glycosyihydrazine (as shown) or other carbonyl-reactivegroups (such as hydrazide, semicarbozide, aminooxyl, etc). Sucholigosaccharides must have one or more phosphate groups attached to theC 6′ position(s) on mannopyranosyl groups (M6P). The oligosaccharidederivatives then react with the carbonyl (aldehyde) groups generated inthe oxidized carbohydrates on glycoproteins to form covalent bondconjugates. FIGS. 2A-2C depict oxidation of the glycoproteins accordingto at least three possible methods. By a first method, sialic acids onglycans are oxidized with a low concentration of sodium periodate (lessthan or equal to 10 mM) to generate the required carbonyl groups. Asecond method is suitable when terminal galactoses exist on the glycans,in which enzymatic oxidation is used. More specifically, galactoseoxidase is used to oxidize the C 6′ hydroxyl group on the galactosegroups. The second oxidation method should not inactivate theglycoprotein. In an alternative embodiment of the second oxidationmethod, sialic acid groups on glycoprotein carbohydrates are removedusing neuraminidase to expose the terminal galactoses, and thengalactose oxidase is used to oxidize the terminal exposed galactoses asdescribed for the first embodiment of the second oxidation method. By athird oxidation method, the hexoses on the glycans are oxidized withrelatively high concentrations of sodium periodate, i.e. with sodiumperiodate having a concentration of greater than about 10 mM and lessthan about 500 mM, to open the vicinal hydroxyl groups of the sugarring. This third oxidation method is potentially harmful to certainglycoproteins that are sensitive to oxidation. To protect theglycoproteins from oxidation of amino acids, reductive agents such asbeta-mercaptoethanol or cysteine or others are added to the oxidationreaction.

In some of the examples infra, a natural phosphorylated oligosaccharide,the phosphopentamannose derived by mild acid hydrolysis ofO-phosphomannan extracted from yeast Hansenula holstii NRRL Y-2448, wasused. This compound has a structure of 6-P-M(alpha 1,3)-M(alpha1,3)-M(alpha 1,3)-M(alpha 1,2)-M (M. E. Slodki, 57 BIOCHIMICA ETBIOPHYSICA ACTA 525 (1962); R. K. Brefthauer et al., 12(7) BIOCHEMISTRY1251 (1973); L. A. Parolis et al., 309 CARBOHYDR. RES. 77 (1998)). Sincethe terminal mannosyl in phosphopentamannose is linked to thepenultimate mannosyl group via alpha 1,3 linkage, this compound exhibitsabout 6 fold less affinity towards the MPRs than the alpha 1,2 linkedmannosyl oligosaccharides (J. Distler et al., 32(15) J. BIOL. CHEM.21687 (1991)). Preferred oligosaccharides for therapeutic purposes willbe those having the terminal and penultimate mannosyl groups linked viaan alpha 1,2 linkage. A trisaccharide bearing a terminal M6P is betterthan a bisaccharide bearing terminal M6P, and a bisaccharide bearingterminal M6P is better than M6P alone (J. Distler et al., 32(15) J.BIOL. CHEM. 21687 (1991); H. Tomoda et al., 213 CARBOHYDR. RES. 37(1991)).

While some of the examples are carried out with the natural product ofphosphopentamannose derivatized with hydrazine, it will be clear to oneskilled in the art that various changes in form and detail can be madewithout departing from the true scope of the invention. For example, theoligosaccharide compounds useful in the present invention include anyoligosaccharides that can be synthesized and derivatized with anychemical group, such as hydrazine, hydrazides, semicarbozide, aminooxyl(L. A. Vilaseca et al., 4(6) BIOCONJUG. CHEM. 515 (1993)) groups, etc.,that can react with carbonyl groups. Total synthesis of variousmannopyranosyl oligosaccharides containing M6P has been reported (O. P.Srivastava and O. Hindsgaul, 155 CARBOHYDR. RES. 57 (1986); O. P.Srivastava and O. Hindsgaul, 52 J. ORG. CHEM. 2869 (1987); O. P.Srivastava and O. Hindsgaul, 161 CARBOHYDR. RES. 195 (1987)).

In addition, numerous biologically active materials are subject tomodification according to the present methods to form novel compoundsand compositions. Bioactive materials that are modified by the presentmethods include glycoproteins, especially lysosomal enzymes isolatedfrom natural sources or produced by recombinant technologies. However,other bioactive materials that are modified by the present methodsinclude antiviral drugs and gene-targeting materials. After modificationaccording to the present methods, the bioactive materials are taken upby target cells through receptor-mediated endocytic pathways. Themodified materials do not lose their biological activity, and thecovalent bonds are stable at neutral pH between 6.5-7.5 for at least fewmonths in solution at 4° C., or indefinitely if lyophilized (J. SinghKralovec et al., 29 CANCER IMMUNOL. IMMUNOTHER. 293 (1989)). Once insidethe cells, however, the covalent bonds in conjugated materials arecleaved into component oligosaccharide derivatives and the biologicallyactive materials by the low pH in the cellular endosomes and lysosomes(pH<5.5) within a relatively short period of time (G. R. Braslawsky etal., 33 CANCER IMMUNOL. IMMUNOTHER. 367 (1991)).

In another embodiment of this invention, other sugar residues that havecognate carbohydrate-binding receptor are modified according to thepresent methods, and oligosaccharide chains on a glycoprotein can beextended. For example, mildly oxidized sialic acid can be extended withmannose or galactose to target the mannose receptor orasialoglycoprotein receptor to achieve tissue or cell-specifictargeting.

In another application of this invention, anti-viral drugs are modifiedwith M6P to enhance their therapeutic efficacy. During viral infection,viral entry also occurs through receptor-mediated endocytosis. Once inthe endosome, the low pH induces fusion of viral membrane with theendosome membrane and releases the viral content to the cytosol to startthe replication cycle. Current anti-viral drugs are mostly lipophilic sothey can pass through the cell membrane and reach cytosol to beeffective; therefore they are general and not cellular compartmentspecific. M6P modification according to the present methods isespecially suitable for developing hydrophilic, cellularcompartment-specific anti-viral drugs. Anti-viral drugs with M6P aretaken up by the cells through MPR-mediated endocytosis to concentrate inendosomes where virus entry occurs, thus subjecting early stage viralinfection to attack by the antiviral compound before viral replication,resulting in improved therapeutic value. A similar approach of involvingcoupling of AZT to mannosylated BSA, which can be taken up by themannose-receptor, has been shown to have higher anti-viral activity thanthe AZT parental drug (G. Molema et al., 34(3) J. MEDICINAL CHEM. 1137(1991)).

In another embodiment of this invention, the methods are used to modifyoligonucleotides useful in gene therapy targeted to correct pointmutation in genes. More specifically, the methods are used to modifyRNA-DNA chimeric oligonucleotides that are used to repair one or twobase pair alterations in the genome of mammalian cells (E. B. Kmiec, 17ADV. DRUG DELIVERY REVIEWS 333 (1995); K. Yoon et al., 93 PROC. NAT.ACAD. SCI. 2071 (1996)). Such a strategy has been used, for example, tocorrect the mutation responsible for sickle cell anemia in vitro (A.Cole-Strauss et al., 273 SCIENCE 1386 (1996)), to mutate the rat factorIX gene and UGT in rat liver in vivo (B. T. Kren et al., 4 NATUREMEDICINE 285 (1998); B. T. Kren et al., 96(18) PROC. NATL. ACAD. SCI.10349 (1999); P. Bandyopadhyay et al., 274 J. BIOL. CHEM. 10163 (2000))and to correct dystrophin in mdx mouse muscle (T. A. Rando et al.,97(10) PROC. NATL. ACAD. SCI. 5363 (2000)). A critical step for successwith this strategy is to deliver the oligonucleotides to target cellswith high efficiency. The percentage of gene conversion correlates withthe efficiency of oligonucleotide delivery, which is enhanced bymodifying polycations or lipsosome with lactose for theasiologlycoprotein receptor on liver hepatocytes (Kren et al. 1998,supra; Kren et al. 1999; supra; Bandyopadhyay et al., supra). Incontrast, for the mdx mouse dystrophin, only the muscle near theinjection site is converted (Rando et al., supra), presumably becauseonly cells nearby the injection site take up the injectedoligonucleotides. Thus, an efficient and general delivery approach ofthe oligonucleotides for a variety of target cells in vivo is especiallyuseful for expanding the application of such gene therapies.Accordingly, the methods described herein permit M6P modification toprovide improved delivery of oligonucleotides to target cells byenhancing MPR-mediated uptake. MPRs are present on a wide variety ofcells in vivo and MPR-mediated endocytic process is as efficient anuptake process as the asialoglycoprotein receptor-mediated endocytosison hepatocytes in liver. PEI/liposome delivery systems employed for theaforementioned oligonucleotides, or the oligonucleotides can be easilymodified with M6P or M6P oligosaccharide derivatives, thus to expand thetarget cell types in vivo for gene-targeted therapy.

The following examples provide illustrative embodiments of theinvention. One of ordinary skill in the art will recognize the numerousmodifications and variations that may be performed without altering thespirit and scope of the present invention. Such modifications andvariations are encompassed within the scope of the invention. Theexamples do not in any way limit the invention.

EXAMPLES Example 1: Synthesis of Phosphopentamannose-HydrazineDerivatives

Phosphopentamannose was prepared from phosphomanan obtained from Dr. M.E. Slodki, Northern Regional Research Laboratory, U.S. department ofagriculture, Peoria, Ill. Phosphopentamannose was prepared essentiallyas described by M. E. Slodki (1962) and has the following structure:6-P-M(alpha 1,3)-M(alpha 1,3)-M(alpha 1,3)-M(alpha 1,2)-M.

100 mg of lyophilized powder of phosphopentamannose was added into aglass tube, to which 3 ml of anhydrous hydrazine was added. The tube wasfilled with nitrogen gas, capped with a tight fitting cap, and wrappedwith parafilm. The reaction was proceeded at room temperature for 6-18hours, after which the hydrazine was evaporated under vacuum while thehydrazine was absorbed through a bottle of sulfuric acid. 2 ml oftoluene was added and removed by a stream of nitrogen gas to get rid ofthe residual hydrazine (Tolvanen and Gahmberg, 1986, supra; Gahmberg andTolvanen, 1994, supra). Phosphopentamannosyl-hydrazine (PPMH) wasdissolved in 2 ml of water and dialyzed against 4 liters of 10 mMphosphate buffer (pH 7.0) overnight at 4° C., after which the sample wascollected and lyophilized.

Example 2: Coupling of Phosphopentamannose-Hydrazine to Avidin

A. Oxidation of Avidin

1 ml of 2.5 mg/ml of avidin (obtained from Sigma or Pierce) wereoxidized with 10 mM sodium periodate in 100 mM sodium acetate (pH 5.6)for 30 minutes at 4° C. in the dark. After which 25 μl of glycerol wereadded and the sample was incubated on ice for 15 minutes to consume theexcess sodium periodate. Samples were then dialyzed overnight against100 mM sodium acetate (pH 5.6) at 4° C. 0.5 ml of 2.5 mg/ml avidinwithout periodate oxidation were processed the same way as untreatedcontrol. Samples after dialysis were collected and stored at 4 or −20°C. until use.

B. Coupling of Phosphopentamannose-Hydrazine to Oxidized Avidin

200 μl of untreated or oxidized avidin (2.5 mg/ml) were mixed with 1 mgof phosphopentamannose-hydrazine dissolved in 20 μl of 100 mM sodiumacetate buffer (pH 5.6) and incubated at 37° C. for 1 hour. The sampleswere dialyzed against 2 liters of CI-MPR binding buffer (50 mMimidazole, 150 mM NaCl, 1 mM EDTA, 0.5 mM MgCl₂, 1 mM beta-glycerolphosphate, 0.025% Triton X-100, pH 7.0) overnight at 4° C. Samples werecollected after dialysis. 10 μl of untreated avidin/conjugated, oxidizedavidin control without conjugation, oxidized avidin/conjugated sampleswere boiled in SDS sample buffer and separated on 12% SDS-gel to see ifthere is any mobility shift. 50 μl of the samples are subjected to theCI-MPR binding test. The remaining samples are stored at −20° C. untiluse.

FIG. 3 is a SDS-PAGE analysis of different avidins before and afterphosphopentamannose-hydrazine conjugation. Lane 1 shows the results foruntreated avidin with conjugation. Lane 2 shows the results for oxidizedavidin without conjugation. Lane 3 shows the results for oxidized avidinwith phosphopentamannose conjugation. Only Lane 3 shows some avidinretardation in migration, indicating conjugation has occurred. As shownin FIG. 3, there is a clear shift of molecular weight in the oxidizedavidin/conjugated sample compare to the untreated avidin/conjugated oroxidized avidin without conjugation controls, indicating that theoxidized avidins are coupled to phosphopentamannose-hydrazine. Themolecular weight shift is about 1-4 kDa, suggesting 1-4phosphopentamannose were coupled to one monomer of avidin.

C. Binding of Unconjugated Avidin and Conjugated Avidin to CI-MPR Column

100 μg of untreated avidin/conjugated (unconjugated) and oxidizedavidin/conjugated in 0.5 ml CI-MPR binding buffer were passed through aCI-MPR column 5 times, and the final passage was collected asflow-through. The column was washed with 8 volumes of binding buffer, analiquot of the final wash was collected, and finally the bound avidinwere eluted with 0.5 ml of 5 mM M6P in binding buffer. 20 μl of the flowthrough, the final wash and the eluted samples were separated on SDS-gelas described for FIG. 3.

FIG. 4 is a CI-MPR binding analysis of untreated avidin and oxidizedavidin conjugated with phosphopentamannose-hydrazine. As is shown inFIG. 4, none of the untreated/conjugated avidin binds to the CI-MPRcolumn, whereas all the oxidized avidin/conjugated binds to the CI-MPRcolumn, indicating the efficiency of coupling ofphosphopentamannose-hydrazine to oxidized avidin is nearly 100%. Theoxidized avidin/unconjugated sample, as the untreated avidin, also doesnot bind to CI-MPR column (data not shown), indicating the couplingprocedure is specific.

Example 3: Conjugation of Phosphopentamannose-Hydrazine toBeta-Glucuronidase does not Inactivate the Enzyme

One major concern about the conjugation is that lysosomal enzymesconjugated in such a way must retain enzymatic activity, preferably fullactivity. While the avidin conjugation result clearly has shown that thecoupling process is highly efficient, whether the coupling processaffect its biological activity is unknown, in particular, avidin is astable protein, not an enzyme. Therefore in the following example,lysosomal enzyme beta-glucuronidase isolated from bovine liver (50,000U/mg, not completely pure, purchased from Sigma) was used.

A. Oxidation

6 mg of beta-glucuronidase were dissolved in 1.5 ml of water, 1.3 ml ofthe material (4 mg/ml) were dialyzed against 100 mM NaAc (pH 5.6)overnight at 4° C. 200 μl of the rest of the sample were kept at 4° C.as water-control.

Of the sodium acetate dialyzed beta-glucuronidase, 0.5 ml were kept asuntreated-dialyzed material, 0.8 ml were oxidized with 10 mM sodiumperiodate at 4° C. for 30 minutes. After which 20 μl glycerol were addedand the sample mixed on ice for 10 minutes to decompose all the excesssodium periodate, then the oxidized material was dialyzed against 1liter 100 mM sodium acetate overnight. 0.4 ml of the sample was kept at4° C. as oxidized-dialyzed control. The other 0.5 ml of sample were usedfor phosphopentamannose-hydrazine coupling.

B. Coupling

3 mg of phosphopentamannose-hydrazine were dissolved in 25 μl of 100 mMNaAc (pH, 5.6) and mixed with 0.5 ml oxidized beta-glucuronidase (4mg/ml) and incubated at 37° C. for 2 hours, the coupled sample wasdialyzed against CI-MPR binding buffer overnight.

C. Enzymatic Activity of Variously Treated Beta-Glucuronidase

To 200 μl of 100 mM p-nitrophenyl beta-glucuronide in 100 mM sodiumacetate (pH 5.0), 15 μl of water as negative control, 15 μl ofbeta-glucuronidase dissolved in water, 15 μl of sodium acetatedialyzed-untreated beta-glucuronidase, 15 μl of oxidized-dialyzedbeta-glucuronidase and 15 μl of oxidizedbeta-glucuronidase+phosphopentamannose-hydrazine were added. Afterincubation at 37° C. for 1 hr, 200 μl of 200 mM glycine (pH 10.4) wereadded. OD of each sample was measured at 400 nm.

The results of one such experiment are described in Table 1.

TABLE 1 Sample OD at 400 nM 15 μl water 0.00 15 μl beta-glucuronidasedissolved in 1.40 water 15 μl untreated NaAc dialyzed beta- 1.37glucuronidase 15 μl oxidized beta-glucuronidase 1.43 NaAc-dialyzed 15 μloxidized beta-glucuronidase plus 1.44 phosphopentamannose

FIG. 5 is a bar graph showing enzymatic activity of beta-glucuronidaseafter different treatments including after conjugation. 1 is H₂Ocontrol; 2, beta-glucuronidase dissolved in H₂O; 3, beta-glucuronidasedialyzed against NaAc (pH 5.6); 4, oxidized beta-glucuronidase dialyzedagainst NaAc, and 5, oxidized beta-glucuronidase conjugated withphosphopentamannose-hydrazine. The results represent an average of threeexperiments and indicate nearly equal beta-glucuronidase activity in allsamples. Thus, the overall procedure did not appear to inactivate thebeta-glucuronidase. This result is expected because the couplingprocedure does not involve the protein backbone, and thus should notaffect the overall protein conformation.

D. CI-MPR Binding and Beta-Glucuronidase Assay

100 μl of untreated beta-glucuronidase (CI-MPR binding buffer dialyzed)and 100 μl of oxidized beta-glucuronidase+phosphopentamannose-hydrazineconjugated (CI-MPR binding buffer dialyzed) were mixed with 400 μl ofCI-MPR binding buffer (pH 7.0). 50 μl of each sample were saved asstarting material for late beta-glucuronidase assay.

450 μl of each sample were passed over a 2 ml CI-MPR column(pre-equilibrated with CI-MPR binding buffer) 5 times. The flow-throughof each sample was saved. The column was washed with 8 volumes of CI-MPRbinding buffer, the last 0.5 ml was saved as final wash. Finally, thecolumn was eluted with 5 mM M6P in CI-MPR binding buffer by passing overthe column 4 times, the eluates were collected as M6P elutions.Therefore each sample has 3 fractions plus the starting materialcontrols. The beta-glucuronidase assay is described below.

To 200 μl of 100 mM p-nitrophenyl glucuronide in 100 mM sodium acetatebuffer (pH 5.0),

30 μl of water,

30 μl of untreated beta-glucuronidase column starting material,

30 μl of untreated beta-glucuronidase flow-through,

30 μl of untreated beta-glucuronidase wash,

30 μl of untreated beta-glucuronidase M6P elution,

30 μl of oxidized beta-glucuronidase+PPMH (imidazole) column startingmaterial,

30 μl of untreated beta-glucuronidase+PPMH flow-through,

30 μl of untreated beta-glucuronidase+PPMH wash,

30 μl of untreated beta-glucuronidase+PPMH M6P elution, were added, thesamples were incubated at 37° C. for 1 hour. 200 μl of 200 mM glycine(pH 10.4) was added to each sample to stop the reaction and OD400 nm wasmeasured.

The results from one such experiment are summarized in Table 2.

TABLE 2 Sample O.D. at 400 nm 30 μl water 0.00 30 μl untreatedbeta-glucuronidase column 1.43 starting material 30 μl untreatedbeta-glucuronidase flow-through 1.36 30 μl untreated beta-glucuronidasewash 0.00 30 μl untreated beta-glucuronidase M6P elution 0.00 30 μluntreated beta-glucuronidase + PPMH 1.41 column starting material 30 μluntreated beta-glucuronidase + PPMH 0.27 flow-through 30 μl untreatedbeta-glucuronidase + PPMH 0.00 wash 30 μl untreated beta-glucuronidase +PPMH 0.06 M6P elution

FIG. 6 is a bar graph comparing the CI-MPR binding results for untreatedand oxidized phosphopentamannose-conjugated beta-glucuronidase. For theuntreated material, nearly 100% of the starting activity was in the flowthrough, nothing was in the final 0.5 ml wash and M6P elution fractions.However, for the oxidizedbeta-glucuronidase+phosphopentamannose-hydrazine sample, only 19% of thestarting activity was in the flow through, nothing in the final 0.5 mlwash and about 5% was in the M6P elution fraction.

For the untreated sample, the total beta-glucuronidase activities in thestarting material and in the flow-through are about equal so there is noclear loss of sample during the column binding. However, for theoxidized/conjugated beta-glucuronidase sample, the total activity of theflow-through and the M6P elution does not add up to the total activityof the starting material. This is not due to the loss of enzymaticactivity by oxidation (FIG. 5), but due to the fact that the oxidizedbeta-glucuronidase conjugated to phosphopentamannose has relatively lowbinding affinity to CI-MPR (J. Distler et al., supra), especially whenone oxidized glycan was conjugated to only onephosphopentamannose-hydrazine due to steric hindrance of the vicinalaldehyde groups. The binding of oligomannosyl phosphate substrates toCI-MPR column has been well characterized (P. Y. Tong et al., 264(14) J.BIOL. CHEM. 1962 (1989); J. Distler et al., supra). The monophosphateform of oligomannosyl substrate binds to CI-MPR column with low-affinitycompare to the bisphosphorylated substrate, with large portion of themonophosphate substrate being eluted during the washing step and therest eluted with M6P, whereas the bisphosphorylated substrate can onlybe eluted with M6P (P. Y. Tong et al., supra). Therefore, the mostlikely possibility is that the oxidized beta-glucuronidase conjugatedwith phosphopentamannose-hydrazine may just behave like the substratewith only one monophosphate and being lost in the first few volumes ofwashing buffer.

Example 4: Preparation of Modified Recombinant Human Acid α-Glucosidase(Neo-rhGAA)

A. Endoglycosidase H Digestion of α-Galactosidase A

One gram of purified recombinant human α-galactosidase A (Genzyme Corp.,Cambridge, Mass.) was reconstituted in 180 ml of deionized water anddialyzed twice against 4 liters of 25 mM acetate buffer (pH 5.6) for 18hours. The dialyzed α-galactosidase A was subsequently mixed with 20 mlof 0.5 M citrate buffer (pH 5.5) containing 1% β-mercaptoethanol.Digestion was performed with 50,000 units of endoglycosidase Hf (NewEngland Biolabs, Beverly, Mass.) at 37° C. for 4 hours or untilcompletion, as determined by SDS-PAGE. Following digestion, the samplewas filtered through a Centriprep-20 column with a molecular weight cutoff of 5000 Da (Millipore, Bedford, Mass.). The filtrate containing thereleased oligosaccharide was collected and dialyzed against threechanges of 4 liters of deionized water at 4° C.

B. Isolation and Derivatization of M6P-Containing Oligosaccharide

M6P-containing oligosaccharides were released from recombinantα-galactosidase A (Genzyme Corp) by digesting with endoglycosidase Hf(New England Biolabs) and purified according to the method of Varki andKornfeld (255 J. BIOL. CHEM. 10847-10858 (1980)) with minormodifications. The dialyzed oligosaccharides were adjusted to 2 mM Trisand then loaded onto a 20 ml QAE-sephadex A column that had beenequilibrated with the same buffer at a flow rate of 1.5 ml/min. Thecolumn was washed sequentially with 2 mM Tris containing 20 mM and 70 mMNaCl, and the M6P-containing oligosaccharides were eluted with 2 mM Triscontaining 200 mM NaCl. The purified M6P-containing oligosaccharides andthe phosphopentamannose were derivatized to glycosylhydrazines using themethod of Tolvanen and Gahmberg. (261 J. BIOL. CHEM. 9546-9551 (1986)).

C. Chemical Conjugation of Derivatized M6P-Containing Oligosaccharidesonto rhGAA

Recombinant human acid α-glucosidase (rhGAA) was dialyzed twice against2 liters of 0.1 M sodium acetate (pH 5.6) for 18 hours at 4° C. Thenucleotide sequence of rhGAA is shown in SEQ ID NO:1 and the amino acidsequence of rhGAA is shown in SEQ ID NO:2. The dialyzed rhGAA (5 mg/ml)was oxidized with 2 mM sodium meta-periodate for 30 minutes on ice.Excess sodium meta-periodate was removed by the addition of 0.5 ml of50% glycerol and incubation on ice for 15 minutes. The oxidized enzymewas then dialyzed against 2 liters of 0.1 M sodium acetate (pH 5.6).Fifty mg aliquots of the oxidized rhGAA were conjugated to 10 mg ofhydrazine-derivatized M6P-containing oligosaccharides or 20 mg ofphosphopentamannose by mixing and incubating at 37° C. for 2 hours.After conjugation, both the M6P- and phosphopentamannose-conjugatedrhGAA samples were dialyzed against 4 liters of 25 mM sodium phosphatebuffer (pH 6.75) containing 1% mannitol and 0.005% Tween-80 for 18 hoursat 4° C. and then sterile filtered. The samples were aliquoted,snap-frozen on dry ice and stored at −80° C. until further analysis.

Example 5: Process for Chemically Conjugating M6P-ContainingOligosaccharides onto rhGAA Did not Affect its Enzymatic Activity

Direct chemical conjugation of oligosaccharides onto a protein backbonevia reductive amidation or maleimide chemistries frequently requiresprolonged incubations at neutral to alkaline pH. These reactionconditions are destabilizing to lysosomal enzymes such as GAA that haveoptimal activities at acidic pH. In order to minimize the inactivationof GAA, a conjugation method was used which employed a condensationreaction between an aldehyde group and a hydrazine to form a hydrozonebond. In this method, M6P-containing oligosaccharides were derivatizedto glycosylhydrazines and then conjugated (at acidic pH) to rhGAA, thesialic acids of which had been oxidized with periodate to aldehydes.Conjugating the M6P-containing moieties directly onto the existingoligosaccharide side chains of rhGAA also confers spacer length thatcould minimize the effect of steric hindrance during receptor binding.

Additionally, conjugation of phosphopentamannose-hydrazine onto rhGAAdid not affect the enzyme's hydrolytic activity (data not shown).Conjugation efficiency was determined to be high and to have occurred onnearly all the rhGAA molecules, as evidenced by an increase in thebinding of the phosphopentamannose-conjugated rhGAA to a CI-MPR column,shown in FIG. 7, where closed circles represent conjugated rhGAA andopen circles represent rhGAA. While only approximately 40% of theoriginal rhGAA bound the CI-MPR column, the column retained greater than90% of the phosphopentamannose-conjugated rhGAA. Therefore, theconjugation process used to modify the oligosaccharides on rhGAA wasefficient and did not measurably alter its activity. However,conjugation with phosphopentamannose did not enhance its uptake into L6myoblasts in vitro when compared to the unmodified enzyme (data notshown). This may be attributed to relatively low-affinity ofphosphopentamannose to CI-MPR.

Example 6: Conjugation of Mono- and Bis-Phosphorylated OligomannoseResidues onto rhGAA Improved its Binding to CI-MPR

Soluble CI-MPR was purified from fetal bovine serum using aphosphopentamannose column according to the method of Li et al. (1GLYCOBIOLOGY 511-517 (1991)). The purified CI-MPR (1 mg) was coupled to1 ml Affigel-15 beads (BioRad) essentially as outlined by themanufacturer. Binding of rhGAA or M6P-conjugated rhGAA to the CI-MPRcolumn was performed as described by Valenzano et al. (270 J. BIOL.CHEM. 16441-16448 (1995)). The M6P content of rhGAA and modified rhGAA(neo-rhGAA) was analyzed using the method described by Zhou et al. (306ANAL. BIOCHEM. 163-170 (2002)). Oligosaccharide profiling of thepurified M6P-containing oligosaccharides following endoglycosidase Hfdigestion of α-galactosidase A was performed according to the method ofTownsend and Hardy. (1 GLYCOBIOLOGY 139-147 (1991)).

To generate a modified rhGAA (neo-rhGAA) with high affinity for theCI-MPR, M6P-containing oligosaccharides were isolated from recombinanthuman α-galactosidase A and conjugated onto rhGAA, as described supra.Recombinant α-galactosidase A was used as a source of theoligosaccharides because analysis of its carbohydrate content, as shownin FIG. 8A, indicated that 30 to 40% of the high-mannoseoligosaccharides are bis-phosphorylated in α-galactosidase A, which isexpected to result in high affinity for the CI-MPR. Phosphorylated highmannose oligosaccharides (both mono- and bis-phosphorylated) werereleased from α-galactosidase A by endoglycosidase H treatment, purifiedover a QAE column and analyzed by dionex column chromatography, shown inFIG. 8B.

Conjugation of the purified mono- and bis-phosphorylatedoligosaccharides onto rhGAA (neo-rhGAA) resulted in an increase in thefraction of enzyme that bound to the CI-MPR column, as shown in FIG. 9,which shows an increase in binding of neo-rhGAA to the CI-MPR column(represented by closed circles), relative to rhGAA (represented by opencircles), and periodate-treated rhGAA (represented by open squares).

As depicted in FIG. 9, approximately 63% of the modified rhGAA(neo-rhGAA) bound to the CI-MPR column compared to approximately 40% forthe unmodified enzyme. The periodate-treated non-conjugated rhGAA (opensquares in FIG. 9) displayed binding characteristics that were similarto that for untreated rhGAA, (open circles in FIG. 9), suggesting thatthe increased binding of neo-rhGAA (closed circles in FIG. 9) wasnot-due to non-specific interactions between the oxidized sialic acidson rhGAA and the CI-MPR column.

A likely reason for a lower than expected fraction of rhGAA containingmono- and bis-M6P (63%, as opposed to >90% forphosphopentamannose-conjugated rhGAA) to bind the CI-MPR column is thatsmaller amounts of the phosphorylated oligosaccharides were used in theconjugation reaction.

Example 7: Conjugation of Mono- and Bis-Phosphorylated OligomannoseResidues onto rhGAA Improved its Uptake into Cells In Vitro

L6 myoblast cells (ATCC) were plated onto 12-well culture dishes andallowed to settle for 24 hours. Prior to the addition of enzyme to thecells, cells were washed once with 3 ml DMEM (Invitrogen, Carlsbad,Calif.). Various forms of rhGAAs in 1 ml of uptake media (DMEMcontaining 1% (v/v) heat inactivated FBS, 25 mM Hepes (pH 6.8), 2.5 mMβ-glycerolphosphate and antibiotics) were added to cells and incubatedat 37° C. for 18 hours. In some of the wells, 5 mM M6P was added toinhibit CI-MPR mediated uptake. After 18 hours, cells were washed twicewith PBS containing 1 mM M6P and then twice more with PBS alone. Cellswere lysed in GAA assay buffer (0.2 M sodium acetate, 0.4 M potassiumchloride, pH 4.3) containing 0.1% Triton X-100 by scraping the cellsfollowed by sonication. Cell lysates were centrifuged at 14,000 g for 10min at 4° C. and the GAA activity in the cleared supernatants wasassayed using the fluorogenic substrate4-methylumbelliferyl-α-D-glucopyranoside. (Sigma Chemical Co., St.Louis, Mo.) (J. L. Van Hove et al., 9 PROC. NATL. SCI. USA 65-70(1996)). Protein content in the cell lysates was determined using themicroBCA kit (Pierce) with BSA as a standard.

Monosaccharide analysis of the neo-rhGAA confirmed that the modifiedenzyme contained higher levels of phosphorylated oligomannose residues.The M6P content was increased from about 0.9 mole M6P/mole of unmodifiedrhGAA, to 2.9 mole M6P/mole of modified rhGAA (neo-rhGAA). Importantly,this increase in M6P-containing oligosaccharides on neo-rhGAA resultedin a significant enhancement in its uptake by L6 myoblasts, as shown inFIG. 10. GAA activity was measured for increasing amounts of rhGAA (opencircles in FIG. 10) or neo-rhGAA (closed squares in FIG. 10). Uptake ofneo-rhGAA approached saturation at 100 nM compared to approximately 500nM for the unmodified rhGAA. This is consistent with an increase in theaffinity of neo-rhGAA for the CI-MPR, presumably because of theconjugation of additional M6P-containing ligands. Uptake was completelyblocked by the addition of excess M6P, confirming that the uptake of theenzyme by the L6 cells was primarily mediated via the CI-MPR (data notshown).

Example 8: Clearance of Glycogen from Pompe Mouse Tissues was Improvedwith Neo-rhGAA

In order to determine whether the improved uptake of modified rhGAA(neo-rhGAA) in vitro in cells correlates with a greater reduction inglycogen storage in vivo in mice, Pompe mice were treated either withneo-rhGAA or unmodified rhGAA. (N. Raben et al. 273 J. BIOL. CHEM.19086-19092 (1998)). Animal experiments were conducted in accordancewith the Guide for the Care and Use of Laboratory Animals (U.S.Department of Health and Human Services, NIH Publication No 86-23).

Four to five month-old Pompe mice were used to evaluate the relativeability of various rhGAAs to reduce glycogen storage in the affectedtissues. (N. Raben et al. supra). Groups of Pompe mice (7 animals/group)were injected via the tail vein with a vehicle (25 nM sodium phosphate,pH 6.5; 1% mannitol; 0.005% Tween-80) and varying doses of rhGAA ormodified rhGAA (neo-rhGAA). Mice were administered three weekly dosesand killed two weeks after the last treatment. Various tissues includingthe heart, diaphragm and skeletal muscles were collected and stored at−80° C. until further analysis. Statistical analysis was performed usingone-way ANOVA followed by a Newman-Keuls test. A probability value ofP<0.05 was considered statistically significant.

The glycogen content in the various muscles of the Pompe mice wasassayed by measuring the difference in the amount of glucose releasedfrom a boiled tissue homogenate following digestion in presence orabsence of Aspergillus niger amyloglucosidase, as described previously.(A. Amalfitno et al., 96 PROC. NATL. ACAD. SCI. USA 8861-8866 (1999)).The glucose levels were assayed using the Amplex Red glucose assay kit(Molecular Probes, Eugene, Oreg.), according to the manufacturer'sinstructions. Bovine liver glycogen (Sigma Chemical Co.) was used as astandard. In some studies, glycogen content was measured using periodicacid Schiff (PAS) staining followed by computer-assistedhistomorphometric analysis (Metamorph) as described previously. (N.Raben et al., 80 MOL. GENET. METAB. 159-169 (2003)). All photography andMetaMorph analyses were performed in a blinded manner.

Approximately 24% and 46% higher enzyme levels were detected in theskeletal muscle and heart tissues, respectively, in animals that wereadministered neo-rhGAA compared to those treated with the unmodifiedrhGAA. Treatment with either form of the enzyme (modified neo-rhGAA orunmodified rhGAA) resulted in a dose-dependent reduction in the glycogenlevels in all tissues examined, as depicted in FIGS. 11A-11D, which isrepresentative of two independent experiments with 7 animals in eachgroup. However, mice treated with neo-rhGAA uniformly displayed agreater reduction in levels of glycogen in all the muscles analyzed, asdepicted in FIGS. 11A-11D.

In the heart, an approximately four to six fold greater reduction inglycogen levels was attained with neo-rhGAA than with rhGAA at both the10 and 20 mg/kg doses (FIG. 11C). Significantly higher reduction inglycogen levels was also observed in the other muscle tissues of animalsthat had been treated with the modified enzyme, as summarized in FIGS.11A, 11B and 11D. An approximately 50% reduction in glycogen level wasattained with 20 mg/kg neo-rhGAA in the quadriceps muscle (FIG. 11A) andto a lesser degree in the triceps (FIG. 11B) and diaphragm (FIG. 11D).In nearly all cases, the efficacy attained with only 20 mg/kg ofmodified rhGAA (neo-rhGAA) was similar to that achieved with 50 mg/kg ofunmodified rhGAA, thereby suggesting that a lower dose of the modifiedrhGAA was sufficient to attain a desirable result. In general, theskeletal muscle tissue was more refractory than the heart and attainedonly a 50-60% reduction in glycogen levels at the 20 mg/kg dose comparedto a nearly a 95% reduction in glycogen levels in the heart. (N. Rabenet al., 80 MOL. GENET. METAB. 159-169 (2003); N. Raben et al., 6 MOL.THER. 601-608 (2002)).

The reduction in glycogen levels observed by biochemical analysis wasconfirmed by histomorphometric assessment of the quadriceps musclesobtained from the same animals. Tissue samples were stained forlysosomal glycogen followed by analysis of tissues by high resolutionlight microscopy (HRLM). Lysosomal glycogen appeared as discreet, purplebeaded structures scattered throughout each myocyte (data not shown).With enzyme treatment, however, these glycogen-containing structuresbecame smaller and fewer in number. The administration of 20 mg/kg ofmodified rhGAA (neo-rhGAA) resulted in about a 54% reduction in thetissue area occupied by glycogen, when compared to the vehicle treatednegative control samples. This reduction was nearly as effective as theadministration of 50 mg/kg of unmodified rhGAA which provided for nearlya 60% reduction, suggesting that neo-rhGAA was 2 to 2.5 times morepotent than rhGAA. The results from one such experiment are summarizedin FIG. 12, which depicts the percentage of area occupied by glycogen inquadriceps muscle sample in vehicle control (A); 10 mg/kg rhGAA (B); 50mg/kg rhGAA (C); and 20 mg/kg neo-rhGAA (D).

Example 9: Modifying rhGAA with Synthetic Bis-M6P Glycans Increased itsBinding to CI-MPR without Affecting its Enzymatic Activity

A. Derivation of Synthetic Bis-M6P-Oligomannose Hydrazide

In order to determine whether synthetic forms of modified rhGAA would beas or more effective than the neo-rhGAA described above, syntheticbis-M6P oligomannose oligosaccharides were conjugated onto rhGAA.

Synthetic bis-M6P glycan was designed based on the in vivo process ofM6P phosphorylation that occurs on naturally occurring high-mannosestructure of lysosomal enzymes. (Kornfeld and Mellman, 5 ANNUAL REVIEWOF CELL BIOLOGY 483 (1989)). Synthetic bis-M6P oligomannose glycan wasderived from the high mannose structure shown in FIG. 13A and was customsynthesized by BioMira (Edmonton, Alberta, Canada).

The middle antennary arm shown in (b) and one terminal mannose in (c)were removed, resulting in bis-M6P oligomannose, which was subsequentlyderivatized with a carbonyl reactive compound, butyryl hydrazide. Theremoval of the middle arm was believed to offer flexibility and improvedpharmacokinetics by reducing competition against mannose receptors onmacrophages and sinusoidal endothelial cells. This derivatized highlyphosphorylated mannose oligosaccharide called bis-M6P-hydrazide, shownin FIG. 13B, was subsequently conjugated onto rhGAA.

B. Chemical Conjugation of Bis-M6P Hydrazide onto rhGAA

Recombinant human GAA (rhGAA) (Genzyme Corp.) was dialyzed twice against4 liters of 0.1 M sodium acetate (pH 5.6) for 18 hours at 4° C.Approximately 10 mg/ml of dialyzed rhGAA was oxidized with 7.5 mM sodiummeta-periodate for 30 minutes on ice. Excess sodium meta-periodate wasremoved by the addition of 50% glycerol and incubation on ice for 15minutes. The oxidized rhGAA was then dialyzed against 4 liters of 0.1 Msodium acetate (pH 5.6). Five hundred milligrams of the oxidized rhGAAwas conjugated to the bis-M6P-hydrazide by mixing and incubating at 37°C. for 2 hours. After conjugation, neoGAA samples were dialyzed threetimes against 4 liters of 25 mM sodium phosphate buffer (pH 6.75)containing 1% mannitol and 0.005% Tween-80 over 24 hours at 4° C. andthen sterile filtered. The samples were aliquoted, snap-frozen on dryice and stored at −80° C. until further analysis.

C. CI-MPR Column Fractionation and In Vitro Cell Uptake

Binding of neo-rhGAA and rhGAA to CI-MPR and in vitro uptake of rhGAAinto L6 myoblast cells was evaluated essentially as described above. Foruptake of GAAs into macrophages, NR8383 macrophage cells (ATCC,Manassas, Va.) were grown in T150 flasks. Prior to the uptake assay,cells were collected, washed once with Kaighan's media (Invitrogen)without serum and resuspended in uptake media (Kaighan's media+1.5%FBS+25 mM Hepes, pH6.8) at a concentration of 0.6-1×10⁶ cells/mi. One mlof cells were aliquoted into microfuge tubes containing 25 nM of rhGAAor neo-rhGAA and to some of the tubes, either 2 mg/ml of yeast mannanwas added to inhibit uptake mediated by mannose receptor, or 5 mM M6P toinhibit uptake mediated by CI-MPR. Uptake was continued for 2 hours at37° C. and cells were harvested by centrifugation, washed twice with PBScontaining 1 mM M6P and 1 mg/ml mannan, and then twice with PBS alone.

All cells were lysed in GAA assay buffer and assayed for GAA activityusing 4-methylumbelliferyl-α-D-glucopyranoside as described above.

As depicted in FIG. 14A, direct conjugation of bis-M6P-hydrazide toperiodate oxidized rhGAA did not affect its specific enzymatic activity.Furthermore, the neo-rhGAA thus generated had increased binding to theCI-MPR column, as depicted in FIG. 14B. In fact, more than 95% ofneo-rhGAA now bound to the CI-MPR column compared to only about 30% ofthe unmodified rhGAA that bound to the column.

Consistent with the increased CI-MPR column binding, monosaccharideanalysis of the neoGAA confirmed that the modified enzyme containedhigher levels of phosphorylated oligomannose residues. The M6P contentwas increased from average of 0.9 mole M6P/mole of unmodified rhGAA toabout 15 mole M6P/mole of neo-rhGAA, which translates to about 7 bis-M6Pglycans conjugated onto the neo-rhGAA.

Furthermore, consistent with a higher affinity for the CI-MPR, theneo-rhGAA also exhibited an improved uptake by L6 myoblasts. As shown inFIG. 15A, uptake of neo-rhGAA approached saturation at about 25 nM(closed squares) compared to approximately 500 nM for the unmodifiedrhGAA (closed circles). Uptake was blocked by the addition of excessM6P, confirming that the uptake of the enzyme by the L6 cells wasprimarily mediated via the CI-MPR. Based on the half maximal value ofuptake, the dissociation constant (kd) of neo-rhGAA to CI-MPR wasestimated to be around 2.5 nM, a value in agreement with the Kd of thenatural bis-M6P oligosaccharides previously reported. (Tong et al., 264J. BIOL. CHEM 7962-7969 (1989)). In contrast, the Kd of the unmodifiedrhGAA to CI-MPR was about 100 nM, also close to the experimentallydetermined Kd for mono-M6P bearing oligosaccharides previously reported.(Distler et al., 266 J. BIOL. CHEM. 21687-21692 (1991)).

Mannose receptors on macrophage cells and sinusoidal endothelial cellsare considered to be responsible for the clearance of glycoproteins invivo by binding to mannose residues. In order to determine whether theaddition of bis-M6P glycans on neo-rhGAA affects its uptake by mannosereceptors, an in vitro cell uptake assay was performed with macrophages.As depicted in FIG. 15B, uptake of rhGAA was barely detectable after 2hours even at 25 nM concentration. Furthermore, inclusion of inhibitorsfor CI-MPR and mannose receptor did not change the GAA activity in thesecells. However, uptake of neo-rhGAA was readily detectable and wasinhibited by M6P, but not by mannan, indicating that the increase inuptake into macrophages was mediated by M6P.

Example 10: Modifying rhGAA with Bis-M6P Hydrazide Resulted in aSignificant Improvement in Glycogen Clearance in Young Pompe Mice

To determine whether the improved uptake and targeting properties ofneo-rhGAA conjugated with synthetic glycan would result in a greaterreduction in glycogen storage, young Pompe mice (5 months of age) weretreated with either neo-rhGAA or unmodified rhGAA. Animal studies wereconducted in accordance with the Guide for the Care and Use ofLaboratory Animals (U.S. Department of Health and Human Services, NIHPublication No 86-23). Groups of Pompe mice (5-7 animals/group) wereinjected via the tall vein with vehicle and varying doses of eitherrhGAA or neo-rhGAA. Mice were administered four weekly doses andsacrificed one week after the last treatment. Various tissues includingthe heart, diaphragm and three skeletal muscles were retrieved andstored at −80° C. until further analysis. Statistical analysis wasperformed using one-way ANOVA followed by a Newman-Keuls test. Aprobability value of P<0.05 was considered statistically significant.

Glycogen content in the various muscles of the Pompe mice was assayed bymeasuring the difference in the amount of glucose released from a boiledtissue homogenate following digestion with or without Aspergillus nigeramyloglucosidase, as described above. Glucose levels from the digestedand undigested sample sets were then assayed using the Amplex Redglucose assay kit according to the manufacturer's instructions. Bovineliver glycogen was used as a standard. In some studies, glycogen contentwas measured using periodic acid Schiff (PAS) staining followed bycomputer-assisted histomorphometric analysis (Metamorph) as describedabove. All photography and MetaMorph analyses were performed in ablinded manner.

Treatment with either form of the enzyme resulted in a dose-dependentreduction in the glycogen levels in all the tissues examined, asdepicted in FIG. 16. At equivalent doses, mice treated with neo-rhGAAuniformly displayed a greater extent of glycogen reduction in all themuscles analyzed, as shown in FIG. 16. In the heart and diaphragm,similar glycogen clearance was achieved with an approximately eight-foldreduced dose of neo-rhGAA relative to rhGAA. For example, 2.5 mg/kg and5 mg/kg of neo-rhGAA achieved nearly the same glycogen reduction as 20mg/kg and 40 mg/kg of rhGAA respectively. Significantly higher reductionin glycogen levels was also observed in skeletal muscles of animals thathad been treated with the modified enzyme. Overall, the dose amount ofmodified rhGAA that was required to achieve a reduction in glycogenlevels similar to that with rhGAA, was about four-fold less than therhGAA in the quadriceps, triceps and psoas muscles examined.

Consistent with previous reports, the heart responded to GAA treatmentbetter than the skeletal muscles which are generally more refractory totreatment, whether by rhGAA or neo-rhGAA. Four weekly doses of 10 mg/kgof neo-rhGAA completely cleared glycogen in the heart to normal levels,whereas 20 mg/kg of neo-rhGAA was required to clear glycogen in thediaphragm and quadriceps. However, for triceps and psoas, at 20 mg/kgdose, some glycogen remained in the muscles, as depicted in FIG. 16.

In an independent study with higher rhGAA and neo-rhGAA doses, it wasobserved that four weekly doses of 20 mg/kg of neo-rhGAA attainedsimilar glycogen clearance as in case of 100 mg/kg rhGAA in the skeletalmuscles, and 40 mg/kg of neo-rhGAA was required to clear glycogen intriceps and psoas muscles to near normal level (data not shown).

The reduction of glycogen observed by biochemical assay was confirmed byhistomorphometric assessment of heart and quadriceps muscle samplesobtained from the same study. By high resolution light microscopy,lysosomal glycogen appeared as discreet, purple beaded structuresscattered throughout each myocyte. With enzyme treatment, theseglycogen-containing structures become smaller and fewer in number. (datanot shown). Administration of 20 mg/kg rhGAA resulted in clear reductionin the number of glycogen granules and percent tissue area occupied byglycogen in the heart when compared to vehicle treated samples; however,less discernible glycogen clearance was observed in quadriceps at thisdose of rhGAA. In both cases, significant glycogen remained in tissues.In contrast, in both heart and quadriceps of Pompe animals treated withneo-rhGAA at this dose, near complete clearance of glycogen wereattained. Occasionally, glycogen granules that appear to be cytoplasmicand resistant to treatment could be seen.

Example 11: Modifying rhGAA with Bis-M6P Hydrazide Resulted in aSignificant Improvement in Glycogen Clearance in Old Pompe Mice

The glycogen storage in old Pompe mice is more resistant to GAAtreatment. This has been attributed, at least in part, to tissue damagein old mice that generally results in less efficient uptake of GAA bytarget muscle cells. To determine if neo-rhGAA would have similarbeneficial effects in old Pompe animals, as in young mice, thirteenmonth old Pompe mice were used for evaluation. About 10 animals/groupwere treated with 40 mg/kg of either rhGAA or neo-rhGAA and the sametissues were harvested and assayed for glycogen content as for theyounger mice.

As shown in FIG. 17, significantly less glycogen were cleared by 40mg/kg of rhGAA in all the tissues examined, as compared to the youngmice. From biochemical analysis of glycogen content, it was estimatedthat only about 80% of the glycogen were cleared in the heart, and only10-40% of the glycogen was cleared from other tissues of old Pompe micetreated with rhGAA. However, despite being more refractory to rhGAAtreatment, glycogen storages were completely cleared from the heart anddiaphragm, >90% in quadriceps and >80% in triceps and psoas when theseold animals were treated with 40 mg/kg of neo-rhGAA.

The increased glycogen clearance by neo-rhGAA over rhGAA in old Pompemice were also confirmed by PAS staining and high resolution lightmicroscopic analysis of representative tissues of the heart andquadriceps (data not shown). Again, as in case of young mice, glycogengranules appeared to be cytoplasmic and resistant to clearance wereoccasionally seen in few muscle cells.

All primary references cited herein are hereby incorporated by referencein their entirety, together with the references contained therein.

The explanations and illustrations presented herein are intended toacquaint others skilled in the art with the invention, its principles,and its practical application. Those skilled in the art may adapt andapply the invention in its numerous forms, as may be best suited to therequirements of a particular use. Accordingly, the specific embodimentsof the present invention as set forth are not intended as beingexhaustive or limiting of the invention.

What is claimed is:
 1. A method of coupling a beta-glucuronidase to anoligosaccharide comprising a phosphorylated mannose, comprising: (a)derivatizing an oligosaccharide comprising a phosphorylated mannose witha compound containing a carbonyl-reactive group; (b) oxidizing abeta-glucuronidase having at least one oligosaccharide to generate atleast one carbonyl group on the at least one oligosaccharide of thebeta-glucuronidase, wherein less than or equal to about 10 mM periodateis used to oxidize one or more sialic acid residues on thebeta-glucuronidase; and (c) reacting the derivatized oligosaccharidewith the oxidized beta-glucuronidase, thereby coupling theoligosaccharide to the beta-glucuronidase.
 2. The method of claim 1,wherein the oligosaccharide comprising the phosphorylated mannose is abiantennary mannopyranosyl oligosaccharide.
 3. The method of claim 2,wherein the biantennary mannopyranosyl oligosaccharide comprisesbis-mannose 6 phosphate (M6P).
 4. The method of claim 1, wherein theoligosaccharide comprising the phosphorylated mannose comprises acompound having the formula 6-P-M_(n)-R wherein: M is a mannose ormannopyranosyl group; P is a phosphate group linked to the C-6 positionof M; R comprises a chemical group containing a carbonyl-reactive group,and n is an integer from 1-15, wherein if n>1, M_(n) are linked to oneanother by alpha (1,2), alpha (1,3), alpha (1,4), or alpha (1,6).
 5. Amethod of claim 4, wherein the oligosaccharide comprising thephosphorylated mannose comprises one of M6P, phosphopentamannose derivedfrom Hansenula holstii O-phosphomannan, and 6-P-M-(alpha 1,2)-M(alpha1,2)-M.
 6. The method of claim 1, wherein the carbonyl-reactive group isa hydrazine, a hydrazide, an aminooxy, or a semicarbazide.
 7. Aconjugate comprising a beta-glucuronidase coupled to an oligosaccharidecomprising a phosphorylated mannose, prepared by the method of claim 1.